• Molecules and Cells
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• v.39 no.11
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• pp.827-833
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• 2016

Regulator of calcineurin 1 (RCAN1) binds to calcineurin through the PxIxIT motif, which is evolutionarily conserved. SP repeat phosphorylation in RCAN1 is required for its complete function. The specific interaction between RCAN1 and calcineurin is critical for calcium/calmodulin-dependent regulation of calcineurin serine/threonine phosphatase activity. In this study, we investigated two available deletion rcan-1 mutants in Caenorhabditis elegans, which proceed differently for transcription and translation. We found that rcan-1 may be required for calcineurin activity and possess calcineurin-independent function in body growth and egg-laying behavior. In the genetic background of enhanced calcineurin activity, the rcan-1 mutant expressing a truncated RCAN-1 which retains the calcineurin-binding PxIxIT motif but misses SP repeats stimulated growth, while rcan-1 lack mutant resulted in hyperactive egg-laying suppression. These data suggest rcan-1 has unknown functions independent of calcineurin, and may be a stimulatory calcineurin regulator under certain circumstances.

• BMB Reports
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• v.48 no.3
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• pp.184-189
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• 2015

Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) is known to protect neurons from neurodegeneration during ischemia/reperfusion injury. We recently reported that ROS-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 in astrocytes and complex formation between caveolin-1 and SHP-2 in response to oxidative stress. To examine the region of SHP-2 participating in complex formation with caveolin-1, we generated three deletion mutant constructs and six point mutation constructs of SHP-2. Compared with wild-type SHP-2, binding of the N-SH2 domain deletion mutant of SHP-2 to p-caveolin-1 was reduced greatly, using flow cytometric competitive binding assays and surface plasmon resonance (SPR). Moreover, deletion of the N-SH2 domain of SHP-2 affected $H_2O_2$-mediated ERK phosphorylation and Src phosphorylation at Tyr 419 in primary astrocytes, suggesting that N-SH2 domain of SHP-2 is responsible for the binding of caveolin-1 and contributes to the regulation of Src phosphorylation and activation following ROS-induced oxidative stress in brain astrocytes.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1397-1401
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• 2005

Bone morphogenetic protein-4 (BMP-4) is a signaling homodimeric molecule that acts as a morphogen to influence cell fate in a concentration-dependent manner. The limited supply of a pure preparation of BMP-4, due to very low level of their expression in vivo, makes it difficult not only to study the biological activities of BMPs, but also to use them as a clinical tool. For a large-scale production of BMP-4, human BMP-4 cDNA was expressed in Chinese hamster ovary (CHO) cells by a recently development vector system, which confers position-independent stable expression of the foreign genes. The CHO cell line expressing recombinant human BMP-4 (rhBMP-4) at the level of $7\;{\mu}g/ml$ could be obtained after stepwise selection with methotrexate. This level of expression is about 70 times higher than those previously reported. The partially processed form of BMP-4 as well as mature form could be detected, when the aliquots of culture media were analyzed by Western blot. The glycosylation pattern and biological activity of the rhBMP-4 were determined by glycosidase treatment and the induction rate of alkaline phosphatase in mouse osteoblastic cells.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.2
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• pp.239-245
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• 2011

This study was conducted to determine the effects of excess vitamin A on alkaline phosphatase (ALP) activity, contents of calcium-binding protein (CaBP), bone gla-protein (BGP) in culture medium and CaBP mRNA expression in chicken osteoblasts in vitro. Osteoblastic cells in the tibia from 1-day-old Arbor Acre broiler chickens were isolated using enzyme digestion. The subconfluenced cells were divided into eight treatments with six replicates in each treatment and cultured in a medium containing either vehicle or different levels of vitamin A (0, 0.2, 0.6, 1.0, 2.0, 5.0, 10.0 and $20.0\;{\mu}g$/ml), and the control received an equivalent volume of ethanol. The incubation lasted 48 h. The results showed that vitamin A down-regulated ALP activity in the culture medium as well as CaBP mRNA expression of osteoblasts in a linear dose-dependent manner (p = 0.124 and p<0.10, respectively), and suppressed the contents of BGP and CaBP in the culture medium in a quadratic dose-dependent manner (p<0.05 and p<0.10, respectively) with increasing addition of vitamin A. The addition of 0-$0.2\;{\mu}g$/ml vitamin A to the culture medium increased ALP activity, BGP and CaBP contents as well as CaBP mRNA expression compared with other groups, but positive effects of vitamin A tended to be suppressed when vitamin A was increased to $1.0\;{\mu}g$/ml, and adverse effects occurred when vitamin A was increased to 10.0-$20.0\;{\mu}g$/ml. These results implied that there was a threshold level of vitamin A inclusion beyond which inhibitory effects occurred, and the mechanism by which overdose of vitamin A reduced bone growth in chickens was probably reduced osteoblastic cell activity, and inhibited expression of CaBP mRNA and CaBP secretion.

• Applied Microscopy
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• v.36 no.4
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• pp.291-297
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• 2006

To evaluate the effects of ingestion of alcoholic drinks on the toxicities of industrial compounds, cyclohexane (CH) was intraperitoneally administrated to rats (1.56g/kg body weight), which had been ingested 15% ethanol for up to 6 weeks,4 times by once a day and every other day. Following the last treatment of ethanol or CH, blood and liver tissues were collected after 4 hours prior to sacrifice of animals. By the injection of CH, liver weight (% of body weight) and xanthine oxidase activity in serum were increased, and glucose-6-phasphatase (G6P) activity in liver was decreased compared to them of control group. The activities of CH metabolizing enzymes, such as cytochrome P450 dependent aniline hydroxylase (CYPdAH) and alcohol dehydrogenase (ADH), were significantly increased by injection of CH, and those activities were the highest in CH-injected group after pretreated with alcohol. Ultrastructurally. both of alcohol treatment and CH injection induced transforming into the smooth-endoplasmic reticulum from rough-endoplasmic reticulum, the those rate was the highest in case of CH-injection after pretreated with alcohol. From these results, it is suggested that alcohol intake on a level without alcoholic degeneration of hepatocytes could enhance the CH metabolism of liver.

• Food Science and Biotechnology
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• v.17 no.2
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• pp.434-437
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• 2008

The aim of this study was to investigate whether Monascus-fermented soybean extracts (MFSE) containing natural estrogen-like compounds such as isoflavones and mevinolins has potential effects on human osteoblast-like SaOS2 cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and alkaline phophatase (ALP) assaies. MFSE exerted biphasic dose-dependent effect; stimulating osteoblastic activity at low concentrations and inhibiting SaOS2 cells viability at high concentrations. At $10^{-8}-10^{-4}\;mg/mL$, MFSE is not only non-cytotoxic but also induced comparatively high ALP activity on SaOS2 cells. ALP activity (%) significantly increased (220.1%, p<0.05) when SaOS2 cells were treated with MFSE at a concentration of $10^{-5}\;mg/mL$, whereas slowly increased (185.6%, p<0.05) in unfermented soybean extracts (UFSE) at $10^{-3}\;mg/mL$. The potentially greater ALP activity of MFSE compared to the UFSE might partially be caused by its mevinolin, which was derived from the soybean during Monascus-fermentation. Our findings indicate that supplementation of MFSE may accelerate the speed of intracellular ALP synthesis by the bone cells when provided at optimal dosages.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2711-2715
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• 2012

Background: Bisphenol A (BPA), an endocrine disrupting chemical, has been suspected to pose carcinogenic risks. However, likely mechanisms are obscure and there are difficulties to estimating its real significance for cancer development. Methods: We therefore studied BPA-induced proteomic alterations in immune organs of ICR mice offspring that were prenatally exposed to BPA (15 and 300 mg/L of drinking water). We performed 2D-gel analyses of samples, considering differences in spleen, exposure levels, sex, and ages. Results: From proteomic analyses, we found various proteins were up- or down-regulated by BPA. Among them, SET, a putative oncogene and inhibitor of phosphatase 2A, was significantly down-regulated in a BPA dose-dependent manner. We also confirmed down-regulation of SET in western blot and real time PCR analyses. From gene network analysis, SET is predicted to communicate with other genes including CYP17, which is involved in biosynthesis and metabolism of sex-hormones. Conclusions: This study provided evidence that SET can be applied as a new biomarker for prenatal BPA exposure and suggests a potential new mechanism of action in that BPA may disrupt CYP17 via SET.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.39 no.4
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• pp.150-155
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• 2013

Objectives: The objective of this study is to determine the adequate concentration and to evaluate the osteogenic potential of simvastatin in human maxillary sinus membrane-derived stem cells (hSMSC). Materials and Methods: Mesenchymal stem cells derived from the human maxillary sinus membrane were treated with various concentrations of simvastatin. The adequate concentration of simvastatin for osteogenic induction was determined using bone morphogenetic protein (BMP-2). The efficacy of osteogenic differentiation of simavastatin was verified using osteocalcin mRNA, and the mineralization efficacy of hSMSCs and simvastatin treatment was compared with alkaline phosphatase and von Kossa staining. Results: Expression of BMP-2 mRNA and protein was observed after three days and was dependent on the concentration of simvastatin. Expression of osteocalcin mRNA was observed after three days in the $1.0{\mu}M$ simvastatin-treated group. Mineralization was observed after three days in the simvastatin-treated group. Conclusion: These results suggest that simvastatin induces the osteogenic potential of mesenchymal stem cells derived from the human maxillary sinus membrane mucosa.

• BMB Reports
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• v.44 no.11
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• pp.735-740
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• 2011

Tunicamycin, an endoplasmic reticulum (ER) stress inducer, specifically inhibits N-glycosylation. The cyclic AMP (cAMP) response element-binding protein H (CREBH) was previously shown to be regulated by UPR-dependent proteolytic cleavage in the liver. On the other hand, the role of CREBH in other tissues is unknown. In the present study, tunicamycin increased the level of CREBH activation (cleavage) as well as mRNA expression in osteoblast cells. Adenoviral (Ad) overexpression of CREBH suppressed BMP2-induced expression of alkaline phosphatase (ALP) and osteocalcin (OC). Interestingly, the BMP2-induced OASIS (structurally similar to CREBH, a positive regulator of osteoblast differentiation) expression was also inhibited by CREBH overexpression. In addition, inhibition of CREBH expression using siRNA reversed the tunicamycin-suppressed ALP and OC expression. These results suggest that CREBH inhibited osteoblast differentiation via suppressing BMP2-induced ALP, OC and OASIS expression in mouse calvarial derived osteoblasts.

• Journal of Applied Biological Chemistry
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• v.42 no.4
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• pp.169-174
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• 1999

Arabidopsis AHL gene contains 4 exons encoding a putative protein highly homologous to the yeast salt-sensitive enzyme HAL2, a 3'(2'),5'-bisphosphate nucleotidase involving in reductive sulfate assimilation. AHL cDNA complemented yeast met22 (hal2) mutant. AHL fusion protein expressed in E. coli exhibited $Mg^{2+}$-dependent, 3'-phosphoadenosine 5'-phosphate (PAP)-specific phosphatase activity. $Li^+,\;Na^+,\;K^+$ and $Ca^{2+}$ ions inhibit the enzyme activity by competing with $Mg^{2+}$ for the active site of the enzyme. The enzyme activity was also sensitive to ${\mu}M$ concentrations of toxic heavy metal ions such as $Cd^{2+},\;Cu^{2+}$ and $Zn^{2+}$, but was not recovered by addition of more $Mg^{2+}$ ions, suggesting that these ions inactivate the enzyme with a mechanism other than competition with $Mg^{2+}$ ions. Inhibition of the AHL enzyme activity may result in accumulation of PAP, which is highly toxic to the cell. Thus, the AHL enzyme could be one of the intial targets of heavy metal toxicity in plants.